|
| Status |
Public on Jun 01, 2010 |
| Title |
Delta dksA Cells Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Delta dksA Cells
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
growth stage: log phase
strain: MG1655 delta lac delta dksA
treatment: No treatment
|
| Treatment protocol |
At OD600~0.3, serine hydroxamate was added to a final concentration of 0.5mg/ml, cells were collected 40 minutes later.
|
| Growth protocol |
Cells were grown in M9 medium supplemented with 0.2% glucose and 0.4% casamino acid at 37℃
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using QIAGEN RNeasy Mini Kit following manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
15 µg of RNA were primed with 2.5 µg of random hexamer at 70°C for 10 min, and reversed transcribed at 50°C for 1 h in the presence of 300 U SuperScript III RTase (Invitrogen), 5xRT Buffer, 10mM DTT, 20U RNaseOut (Invitrogen) and 500 µM each dATP, dCTP, dGTP, with 200 µM dTTP, 600 µM aa-dUTP. cDNA samples were incubated with Cy3 (reference) or Cy5 (sample) for 2hrs.
|
| |
|
| Channel 2 |
| Source name |
pooled reference RNA
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
growth stage: log phase
strain: MG1655 delta lac + MG1655 delta lac delta dksA
treatment: Pooled RNA from mixture of the same amount of wt, wt+SHX, dksA, dksA+SHX RNA samples
|
| Treatment protocol |
At OD600~0.3, serine hydroxamate was added to a final concentration of 0.5mg/ml, cells were collected 40 minutes later.
|
| Growth protocol |
Cells were grown in M9 medium supplemented with 0.2% glucose and 0.4% casamino acid at 37℃
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using QIAGEN RNeasy Mini Kit following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
15 µg of RNA were primed with 2.5 µg of random hexamer at 70°C for 10 min, and reversed transcribed at 50°C for 1 h in the presence of 300 U SuperScript III RTase (Invitrogen), 5xRT Buffer, 10mM DTT, 20U RNaseOut (Invitrogen) and 500 µM each dATP, dCTP, dGTP, with 200 µM dTTP, 600 µM aa-dUTP. cDNA samples were incubated with Cy3 (reference) or Cy5 (sample) for 2hrs.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed following Agilent Two-Color Microarray-Based Prokaryote Analysis Protocol.
|
| Scan protocol |
Scanned on a GenePix 4000B Scanner (Axon Instruments).
|
| Description |
Biological replicate 2 of 3. MG1655 delta lac delta dksA cells untreated
|
| Data processing |
GenePix Pro (v 6.1) was used for background subtraction and normalization.
|
| |
|
| Submission date |
Jan 05, 2010 |
| Last update date |
Jan 05, 2010 |
| Contact name |
Jue D. Wang |
| Organization name |
Baylor College of Medicine
|
| Department |
Molecular and Human Genetics
|
| Lab |
Wang
|
| Street address |
1 Baylor Plaza, Rm T932
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL9855 |
| Series (1) |
| GSE19742 |
Expression profiling of E.coli K-12 wild-type vs. delta dksA cells upon amino acid starvation |
|
